Lecture 22: Glycolysis Part I

What regulates metabolic pathways?

the energy-change of the cell

What happens when the ratio of ATP to ADP is high?

a biosynthetic pathway occurs in

Does glycolysis require or use oxygen?

nope!

Where does glycolysis take place?

the cytoplasm

What are the major pathways of glucose utilization?

• extracellular matrix and cell wall polysaccharides

  • occurs through the synthesis of structural polymers

• ribose-5-phosphate

  • occurs through the oxidation via pentose phosphate pathway

• glycogen, starch, and sucrose

  • occurs through storage

• pyruvate

  • occurs through oxidation via glycolysis

What are the major carbohydrates in the human diet?

• starch

• sucrose

• lactose

• fructose

• glucose

What is the structure of starch?

long polymers of glucose linked by linear a(1→4) glycosidic bonds and a(1→ 6) branches; branched are less prevalent in starch than in glycogen

Which carbon is the anomeric carbon?

C1

What is the process for carbohydrate digestion (up to the intestines)?

1. starches are broken down by salivary a-amylase in the back of the throat

2. starch is further broken down by pancreatic a-amylase

3. a-maltose, a-isomaltose, and trisaccharides are produced

4. sucrose is broken down by sucrase on the brush border of the intestinal epithelial cells

5. lactose is broken down by lactase on the surface of the brush border of the intestinal epithelial cells into glucose and galactose

Which bond does salivary and pancreatic a-amylase catalyze the hydrolysis of?

the a(1→ 4) glycosidic bonds

What is sucrose?

a disaccharide of glucose linked to fructose in a a(1→ 2) linkage

What does sucrase-isomaltose complex deficiency result in? Which people is it highly prevalent in?

• results in an intolerance of ingested sucrose

• highly prevalent in the Inuit people

What is the treatment for sucrase-isomaltase complex deficiency?

dietary restriction of sucrose and enzyme replacement therapy

What is lactose?

a disaccharide of galactose and glucose through a B(1→ 4) linkage

What is responsible for lactose intolerance?

the absence of intestinal lactase

How many people does lactose intolerance affect?

¾ of the world’s adults

What are the treatments for lactose intolerance?

• reduce consumption of milk while eating yogurts and cheeses

• eat green vegetables such as broccoli to ensure adequate calcium intake

• use lactase-treated products

• take a lactase pill prior to eating

Describe SGLT1 (sodium-glucose linked transporter 1).

• present on the apical border of intestinal epithelial cells

• is a symporter that transports glucose, galactose, and Na⁺ against a concentration gradient using energy provided by an electrochemical gradient of sodium

Describe GLUT2 (facilitated glucose transporter).

• transports glucose down its concentration gradient

• present on the basolateral side

• high capacity (V_max)

• low affinity (high K_m) for glucose

• expressed in hepatocytes

• insulin independent

Describe GLUT5 (facilitated fructose transporter).

present on the apical border of intestinal epitherlial cells

What does SGLT1 deficiency cause?

glucose and galactose malabsorption

What does GLUT5 deficiency cause?

fructose malabsorption/dietary fructose intolerance

Explain how glucose gets transported into cells.

1. At the portal (5-20 mM), the GLUT2 receptor allows glucose to enter the liver, pancreatic islets, and intestines

2. B cells secrete insulin in response to a higher blood glucose level

3. glucose uptake is stimulated by muscle and adipose tissue 

4. At the peripheral, GLUT4 (insulin dependent) allows glucose to enter muscle and adipose tissue

5. GLUT4 won’t be present on the membrane if insulin isn’t present

Describe GLUT4.

• insulin dependent

• high affinity for glucose

• low K_m

True or False: Insulin stimulates movement of GLUT4 to the plasma membrane from intracellular vesicles.

true

1 glucose leads to how many pyruvate?

2 pyruvate

Why glucose for glycolysis?

• most stable hexose

  • equatorial substituents

• low potential for non-enzymatic protein glycosylation

  • unreactive pyranose (hemiacetal)

• readily polymerized for storage

• when the open form of the sugar is needed, enzymes will open the ring system

  • this process doesn’t occur in other hexoses

What 2 stages does glycolysis occur in?

1. preparatory phase

2. pay off phase

What are the reactants and products for the prepatory phase?

• reactants: glucose + 2 NAD⁺ + 2 ADP + 2 P_i

• products: 2 pyruvate + 2 NADH + 2 H⁺ + 2 H₂O

What are the steps in the preparatory phase?

1. Glucose is phosphorylated at C6 to produce glucose 6-phosphate

2. Isomerization moves the carbonyl from C1 to C2 to form fructose 6-phosphate

3. OH group on C1 is phosphorylated to produce fructose 1,6-biphosphate

4. carbonyl group at C2 facilitates a C-C bond cleavage to produce dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (2 phosphorylated, 3-C sugars)

What does phosphorylation of glucose ensure?

ensures that pathway intermediates remain in the cell

True or False: Steps 1-3 have asymmetrical products at C6, and step 4 has a pseudo-symmetrical product at C6.

true

Which steps in the preparatory phase require ATP?

steps 1 and 3

How many ATP molecules does the energy investment phase require?

2 ATPs

What is important to know about step 1?

• reactant: glucose

• product(s): glucose 6-phosphate

• enzyme: hexokinase

• reversible or irreversible: irreversible

• ATP or no ATP: ATP

• favorable or unfavorable: favorable/exergonic

• cofactors: Mg²⁺ (helps shield some of the electrostatic charge)

• cost or no cost: costs 1 ATP

• other notes: first priming phase

Do you know the hexokinase reaction?

yes!

True or False: Mg²⁺ coordination makes phosphorus more electrophilic (tendency to attract or acquire electrons).

true

Describe the hexokinase conformational change.

• glucose binds first

• the cleft closes and dehydrates the active site—preventing the nucleophilic attack by water and nonproductive ATPase action

• non-polar active site adds electrostatic free energy to ATP—favoring phosphate-donation

Why should a phosphoryl group be added to glucose?

• the phosphoryl group traps glucose inside of the cell

• acts as a handle for enzyme recognition and provides increased binding free energy

• acts as a progenitor for capturing high-energy intermediates

Describe hexokinase I.

• low K_m

• low V_max

• permits efficient phosphorylation and subsequent metabolism of glucose even at low concentrations

What is hexokinase I inhibited by?

glucose-6-P

What does inhibition of hexokinase I by G-6-P prevent?

prevents it from tying up all of the intracellular P_i in the form of G-6-P

Where is hexokinase IV (glucokinase) expressed?

hepatocytes (liver) and pancreatic B cells

Describe glucokinase/hexokinase IV.

• high K_m

• high V_max

• doesn’t get saturated

• starts to act only when glucose is higher

What indirectly inhibits glucokinase/hexokinase IV?

fructose 6-P

What is the most likely way to divide hexose?

through an aldol cleavage

What is important to know about step 2?

• reactant: glucose 6-phosphate

• product(s): fructose 6-phosphate

• enzyme: phosphoglucose isomerase or glucose 6-PO4 isomerase

• ATP or no ATP: no ATP

• favorable or unfavorable: slightly unfavorable/endergonic

• cofactors: no cofactors

• cost or no cost: no cost

• other notes: converts an aldehyde to a ketone; enzyme acts on the open form of the sugar

What is the process for step 2?

1. binding and opening of the ring

2. proton abstraction by active-site Glu B₁

3. formation of a C=C double bond

4. electrons from carbonyl form an O-H bond with the hydrogen ion donated by B₂

5. an electron leaves the C=C bond to form a C-H bond with the proton donated by B₁

6. B₂ abstracts a proton—allowing the formation of a C=O bond

7. cis-enediol formation

8. general acid catalysis by same Glu facilitates formation of fructose 6-phosphate

9. dissociation and closing of the ring

True or False: The hydrogen that needs to be extracted on glucose-6-phosphate has a pK_a of 20.

true

What is important to know about step 3?

• reactant: fructose 6-phosphate

• product(s): fructose 1,6-bisphopshate

• enzyme: phosphofructokinase-1 (PFK-1)

• reversible or irreversible: irreversible

• ATP or no ATP: ATP

• favorable or unfavorable: favorable/exergonic

• cofactors: Mg²⁺ (helps shield some of the electrostatic charge)

• cost or no cost: costs 1 ATP

• other notes: rate-limiting step; first committed step

What does a PFK-1 deficiency cause?

hemolytic anemia

What is hemolytic anemia?

a condition where red blood cells are destroyed faster than the body can replace them—leading to a shortage of healthy red blood cells

What is PFK-1 allosterically inhibited by?

ATP

Where does ATP bind to PFK-1 to allosterically inhibit it?

binds at a 2nd site away from the catalytic site 

What reverses PFK-1 inhibition by ATP?

AMP (PFK-1 is very sensitive to AMP regulation)

During vigorous exercise, what do cells do to quickly meet the demand for ATP?

cells use the adenylate kinase reaction: ADP + ADP → / ← ATP + AMP

What does the activity of adenylate kinase lead to?

the regeneration of some ATP but an increase in other AMP levels

Elevated ATP levels can what?

inhibit glycolysis

True or False: When citrate levels are high, F-6-P isomerizes back into G-6-P, which inhibits hexokinase and shunts excess G-6-P towards glycogen synthesis.

true

What is an allosteric activator of PFK-1?

fructose 2,6-biphosphate

What is important to know about the fructose 2,6-biphosphate velocity curve?

• the sigmoidal dependence of velocity on the substrate concentration becomes hyperbolic in the presence of fructose 2,6-biphosphate—indicating that more of the enzyme is active at lower substrate concentrations in the presence of fructose 2,6-biphosphate

• ATP, acting as a substrate, initially stimulates the reaction

• as the concentration of ATP increases, it acts as an allosteric inhibitor

• the inhibitory effect of ATP is reduced by fructose 2,6-biphosphate, which makes the enzyme less sensitive to ATP inhibition